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LSP1-Myosin1e Bi-Molecular Complex Regulates Focal Adhesion Dynamics

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LSP1-Myosin1e Bi-Molecular Complex Regulates Focal Adhesion Dynamics bioRxiv preprint doi: https://doi.org/10.1101/2020.02.26.963991; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. LSP1-myosin1e bi-molecular complex regulates focal adhesion dynamics and cell migration Katja Schäringer1*, Sebastian Maxeiner1*, Carmen Schalla1, Stephan Rütten2, Martin Zenke1 and Antonio Sechi1 1Institute of Biomedical Engineering, Dept. of Cell Biology, RWTH Aachen University, Pauwelsstrasse, 30, D-52074 Aachen, Germany 2Electron Microscopy Facility, Institute of Pathology, RWTH Aachen University, Pauwelsstrasse, 30, D-52074 Aachen, Germany *equal contribution Corresponding author: Email: [email protected] Telephone: +49 241 8085248 Running head: LSP1-myosin1e complex regulates cell migration. Keywords: Actin cytoskeleton remodelling, focal adhesions, cell motility, macrophages. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.26.963991; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Several cytoskeleton-associated proteins and signalling pathways work in concert to regulate actin cytoskeleton remodelling, cell adhesion and migration. We have recently demonstrated that the bi-molecular complex between the leukocyte-specific protein 1 (LSP1) and myosin1e controls actin cytoskeleton remodelling during phagocytosis. In this study, we show that LSP1 down regulation severely impairs cell migration, lamellipodia formation and focal adhesion dynamics in macrophages. Inhibition of the interaction between LSP1 and myosin1e also impairs these processes resulting in poorly motile cells, which are characterised by few and small lamellipodia. Furthermore, cells in which LSP1-myosin1e interaction is inhibited are typically associated with inefficient focal adhesion turnover. Collectively, our findings show that the LSP1-myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodelling and focal adhesion dynamics required for cell migration. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.26.963991; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction A large library of actin-associated proteins steer nucleation, cross-linking, capping and elongation of actin filaments. The precise spatial and temporal co-ordination of these functions is fundamental for the movement of cells that is required for many biological events ranging from organ development to tissue repair. The importance of actin cytoskeleton dynamics is emphasised by the onset and progress of diseases due to cells lacking or expressing mutated variants of actin-associated proteins (Mathieson, 2012; Ramaekers and Bosman, 2004). In spite of several studies, the functions of some actin-associated proteins have not been well defined. One of such proteins is the leukocyte-specific protein 1 (LSP1). LSP1 is expressed in several cell types of the immune system such as T-cells, B-cells, macrophages and neutrophils. It is also expressed in myeloid and lymphoid cell lines and, despite its name, in endothelial cells (Jongstra et al., 1994; Jongstra et al., 1988; Jongstra-Bilen et al., 2000; Kadiyala et al., 1990; Liu et al., 2005; Maxeiner et al., 2015; Palker et al., 1998). The amino-terminal half of LSP1 incorporates Ca2+-binding sites and a coiled-coil region (Jongstra et al., 1988; Klein et al., 1989), suggesting that Ca2+ signalling and dimerization could regulate LSP1 function. The carboxy-terminal half incorporates a caldesmon-like region having a weaker F-actin-binding activity (Zhang et al., 2000; Zhang et al., 2001) and two villin headpiece-like sequences, which primarily mediate the interaction of LSP1 with F-actin (Klein et al., 1990; Wong et al., 2003; Zhang et al., 2001). We have demonstrated that the carboxy-terminal half of LSP1 directly interacts with the SH3 domain of the molecular motor myosin1e through the non- canonical SH3-binding site AGDMSKKS (Maxeiner et al., 2015). These studies suggest that LSP1 may be involved in the regulation of actin cytoskeleton architecture and dynamics. Indeed, the actin-binding activity of LSP1 is required for the formation of the long, actin-rich cell projections that develop in a wide-ranging variety of cells, which overexpress LSP1 (Howard et al., 1998; Miyoshi et al., 2001; Zhang et al., 2001). 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.26.963991; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. We have provided a direct evidence that LSP1 regulates actin cytoskeleton dynamics. We found that LSP1 localisation and dynamics at internalisation sites during Fcg receptor-mediated phagocytosis, a process that depends on actin dynamics, spatially and temporally overlap with that of the actin cytoskeleton (Maxeiner et al., 2015). Moreover, in LSP1-deficient macrophages and in macrophages in which LSP1- myosin1e or LSP1-actin interactions are inhibited, Fcg receptor-mediated phagocytosis is severely reduced (Maxeiner et al., 2015). Given the modulation of actin dynamics by LSP1, it is not surprising that LSP1 has been implicated in the regulation of migration of several cell types including neutrophils, dendritic cells and T-cells (Coates et al., 1991; Howard et al., 1994; Howard et al., 1998; Hwang et al., 2015; Jongstra-Bilen et al., 2000; Koral et al., 2015; Li et al., 2000; Petri et al., 2011). Although these studies clearly show that LSP1 is involved in the regulation of actin cytoskeleton structural organisation and dynamics, the molecular mechanisms underlying the function of this actin-associated protein are still poorly characterised. Current evidence shows that LSP1 is phosphorylated at serine and threonine sites (Carballo et al., 1996; Huang et al., 1997; Jongstra-Bilen et al., 1990; Matsumoto et al., 1995a; Matsumoto et al., 1993; Wu et al., 2007). In lymphocytes, LSP1 is phosphorylated by protein kinase C (PKC) (Carballo et al., 1996; Matsumoto et al., 1995b; Matsumoto et al., 1993), whereas in neutrophils stimulated with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), LSP1 is phosphorylated by the mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) (Huang et al., 1997; Wu et al., 2007). Notably, PKC-dependent phosphorylation of LSP1 decreases its localisation with the plasma membrane and the actin cytoskeleton (Matsumoto et al., 1995b; Miyoshi et al., 2001). By contrast, LSP1 phosphorylated by MK2 results in the accumulation of phosphorylated LSP1 at the leading edge of neutrophils (Wu et al., 2007). The importance of the interaction between kinases and LSP1 is further supported by the observation that LSP1 targets 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.26.963991; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. proteins of the ERK/MAP kinase pathway to the actin cytoskeleton (Harrison et al., 2004). Thus, it is plausible that the localisation of LSP1 to actin-rich structures depends on its phosphorylation status and can be regulated by diverse kinases and signalling pathways. Regardless the positive or negative regulation of cell migration, it is unquestionable that LSP1 controls this important biological process. By contrast, very little is known about the molecular mechanisms underlying this LSP1 function. For instance, it has been shown that LSP1 participates in a complex with WASP and the Arp2/3 complex (Prasad et al., 2012), two important regulators of actin filament nucleation. Furthermore, LSP1 can also be found in a complex together myosin IIA and one of its regulators, the myosin light chain kinase (Cervero et al., 2018). Since LSP1 does not directly interact with WASP, the Arp2/3 complex and myosin IIA, it is likely that LSP1 is recruited to these complexes via its interaction with F-actin. Notably, we have demonstrated that LSP1 binds to the SH3 domain of myosin1e and that this bi- molecular complex is essential for efficient actin cytoskeleton dynamics during Fcg receptor-mediated phagocytosis (Maxeiner et al., 2015). In this study, we have added another piece to the puzzle describing the modus operandi of LSP1. We have demonstrated that the interaction of LSP1 with myosin 1e is essential for efficient focal adhesion dynamics and zyxin kinetics at these locations. The LSP1-myosin1e binary complex also regulates lamellipodia formation and dynamics. Consequently, interfering with LSP1-myosin1e interaction impaired cell migration. 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.26.963991; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Materials and Methods Cell culture Wild type and genetically modified J774 macrophage cell lines were grown in DMEM supplemented with 10% fetal calf serum (FCS), 4 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin.
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